LOCUS       sequence-343881-        5922 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pBM23 contained pyrF and mini-CRISPR..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5922)
  AUTHORS   Lin L, Chen J, Mitra R, Gao Q, Cheng F, Xu T, Zuo Z, Xiang H, Han J
  TITLE     Optimising PHBV biopolymer production in haloarchaea via 
            CRISPRi-mediated redirection of carbon flux.
  JOURNAL   Commun Biol. 2021 Aug 25;4(1):1007. doi: 10.1038/s42003-021-02541-z.
  PUBMED    34433872
REFERENCE   2  (bases 1 to 5922)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Commun 
            Biol."; date: "2021-08-25"; volume: "4(1):1007. doi"; pages: " 
            10.1038/s42003-021-02541-z"
COMMENT     Sequence Label: pBM23-pyrF-NT
FEATURES             Location/Qualifiers
     source          1..5922
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(199..218)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(200..218)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(240..262)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    251..267
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(275..281,282..299,300..305))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    320..341
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(458..475)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     CDS             671..1465
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             671..1465
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(725..744)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1335..1354
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     rep_origin      complement(1649..2237)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1729..1748)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2551..3342,3343..3411))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGAQVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEEDKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     3174..3193
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3412..3516)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3584..3602
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3640..3662)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3762..3781
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3975..3997
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     promoter        4018..4036
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     4018..4035
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(4667..4695)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
ORIGIN
        1 gctttcagtc gggcggtggc ggcccacagc agcgcgaaac cggcgggttc ggtgcagggc
       61 cacgacgtgg tccccgagaa ggccccggtg caggacggtt cgggcgcgcc ggacgacagg
      121 gtggaccgca aagtggcgca cgtactgaaa gggcgacacg cccgggtcta gactgcagct
      181 cgaggcatgc aagctttgcc ctatagtgag tcgtattaca attccatcca tagctgtttc
      241 ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt
      301 gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc
      361 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
      421 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct
      481 cggtcgttcg gctgcggcga gcggtatcag ctcactctgg acagcaagcg aaccggaatt
      541 gccagctggg gcgccctctg gtaaggttgg gaagccctgc aaagtaaact ggatggcttt
      601 cttgccgcca aggatctgat ggcgcagggg atcaagctct gatcaagaga caggatgagg
      661 atcgtttcgc atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga
      721 gaggctattc ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt
      781 ccggctgtca gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct
      841 gaatgaactg caagacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg
      901 cgcagctgtg ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt
      961 gccggggcag gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc
     1021 tgatgcaatg cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc
     1081 gaaacatcgc atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga
     1141 tctggacgaa gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgag
     1201 catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat
     1261 ggtggaaaat ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg
     1321 ctatcaggac atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc
     1381 tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta
     1441 tcgccttctt gacgagttct tctgaattat taacgcttac aatttcctga tgcggtattt
     1501 tctccttacg catctgtgcg gtatttcaca ccgcggatct ggcggtaata cggttatcca
     1561 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     1621 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     1681 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     1741 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     1801 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     1861 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     1921 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     1981 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     2041 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg
     2101 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     2161 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     2221 gaaaaaatcg atctcaagaa gaacctttga tcttttctac ggggcgcaga aaaaatgcat
     2281 ctcaagaaga acgtttgatc ttttctacgg gcgcagaaaa aatcgttctc aagaagatgc
     2341 tttgatcttt tctacggggc gcagaaaaaa acgatctcaa gaagaaggtt tgatcttttc
     2401 tacgggatct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
     2461 atcaaaaagg ctcttcacct agagcctttt aaattaaaaa tgaagtttta aatcaatcta
     2521 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
     2581 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     2641 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg
     2701 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     2761 tggtcctgca actttatcct cctccatcca gtctattaat tgttgccggg aagctagagt
     2821 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
     2881 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     2941 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
     3001 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     3061 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     3121 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
     3181 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
     3241 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactt gtgcacccaa
     3301 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
     3361 aaatgccgca aaaaatggga taagcgcgac acggaaatgt tgaatactca tactcttcct
     3421 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     3481 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc
     3541 tgacgtctaa gaaaccatta ttatcatgac attaacctat aaaaataggc gtatcacgag
     3601 gccctttcgt ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca tgcagctccc
     3661 ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc gtcagggcgc
     3721 gtcagcgggt gttggcgggt gtcggggctg gcttaactat gcggcatcag agcagattgt
     3781 actgagagtg caccatatgc ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg
     3841 catcaggcgc cattcgccat tcaggctgcg caactgttgg gaagggcgat cggtgcgggc
     3901 ctcttcgcta ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat taagttgggt
     3961 aacgccaggg ttttcccagt cacgacgttg taaaacgacg gccagccaat tgaagctatt
     4021 taggtgacac tatagaatac gagctcggat ccatggtacc cgggcgtgtc gcccttgcgt
     4081 acatcctcga ccagccgata ctccgtgctg tcgcagtgga ccttggtatc cccgagtcag
     4141 cgacaaatac ggccgtgagt acctcgtatg ccgccttctc gattatcgcg gcgggggcaa
     4201 cctaccgttc cgggtggatt agcgaccgac tcggcgtccg acggtggttc ctcgtctcgc
     4261 cgcttctcgt cgccgtgtcc tacgccgtgc tcccaatctt cggtccactg gcgttcccgg
     4321 tatttgccgt cgttcgcggc gtaaacaacg tctcgggcgt cctcggaaac cagtacatca
     4381 acgaccgtgt cgactcggtt gggcgcgcaa ccgtcctctc cgccgccggg atgctctact
     4441 cgctcgccgt cgttccgttc gaactcgcgg gtggtgtcgt cgccgacgtg gtctctccaa
     4501 ccggcgcact cgccctattc ggcggggttc ttgcggtcgg tgcaggcgtg ttgtgggttg
     4561 tcgaacgccc aatctgaaat ggtgtcgaag ggaacatata tgttactgca ggtacaacac
     4621 cgagttagga ggttacagac gaaccctagt tgggttgaag cctggagttg tcccaattct
     4681 tgttgaatta gatggtgtta cagacgaacc ctagttgggt tgaagctttt ttttgttttc
     4741 ttgtgtgcgg ttcccttgcg tacagttccc agcgaccttc ccggctgcga cgaagggaaa
     4801 accccttaca tacggtcgtc gcactcgcgg gcaatgggct tctttgacga ccttcaggac
     4861 cgcatcgaga aggtggacag tgtcgtctcg gtcggactcg acgccgacct cgaccggatt
     4921 cccgaacacc tccgcgacga ggacctgccg cggtgggcgt tcaaccgccg catcatcgac
     4981 gcgacgcacg aacacgcggc ggcctacaag ccgaacgcgg ccttctacga ggacgaagac
     5041 ggctggcgct cgctccgcga gaccatcgcc tacgcacacg gaaaggatgt tcccgtcctc
     5101 ctcgacgcca agcgcgccga catcggcaac acgacgcgac agtacgcgaa cctgcttgac
     5161 gacgccgatg ccatcaccgt caacccctac atgggccgcg attccatcca gccgttcctc
     5221 gcccgcgagg acaagggcgt gttcgtcctc tgtcgcacct ctaacccggg cagcgcggac
     5281 ctccagtcgc tcgaactcgc ctccggagaa cccctgtacg agcgcgtcgc ggcgctctgc
     5341 gacctctgga acgaaaacga caacgtcgga ctcgtcgtcg gcgcgaccgg ccccgaagag
     5401 ttggaagacc tccgcgagca ggtccccgac ctgccgtttc tcgtccccgg cgtcggcgca
     5461 cagggtggcg atgccgaggc ggcagtcgag tacggactgg cagacggcgt cggtctcgtg
     5521 aactcctcac gcggcatcat cttcgccggc gaagaccagg gagaacactt cgacaaggct
     5581 gcgggacagg cggcgaagcg actcaaagag cggctgaacc agtaccggta agtctgcgga
     5641 aatcacagac tggcactgga ctgtaccgct ctcggcggga cgtttaccag cgtgcgtcac
     5701 caactacgtc cgtggaccga gaccgactta tcatgggcct cgcggcggtg ttcgcgggta
     5761 ttaccatgct cctcgtcgtg ttggcattcg cctaccgaca gctgttgttg ttgttcgtag
     5821 ctgtgccctt cggcgcgacg acgtacttca tgtactcgga cgtgagcggc cggttcgaag
     5881 aacggtttag acggcgagtt caggcggatg ctggcggggt cg
//
